PT1931998E - A method for quantification of allergens - Google Patents

Description

1

Description " Allergen quantification method "

FIELD OF THE INVENTION The present invention relates to the field of allergen quantification.

BACKGROUND OF THE INVENTION

Allergens are antigenic molecules that induce allergic responses and the production of IgE antibodies in humans. They are used in the diagnosis and treatment of allergy, for example, in immunotherapy with allergens, in the form of allergen vaccines. The source materials of allergens to which humans are exposed, such as food, pollens or fecal mite particles, naturally occur as complex mixtures of major or minor allergens. The major allergens are allergens to which most allergic patients respond to this source. However, it appears that any protein is a potential allergen, as more and more allergens are identified as knowledge in the area expands. Thanks to the complexity of the allergen sources, the amino acid sequences of various allergens were initially deduced from nucleotide sequences derived from cDNA. Cloning of genes encoding allergens has shown that most allergens are heterogeneous and that they can occur under the form of mixtures of isoallergens and variants. Alignment of the amino acid sequence of homologous allergens and isoallergens demonstrated that these can be identified by and / or divided into constant and variable region sequences. The constant region amino acid sequences are unique to the species, whereas the variable region amino acid sequences are unique to each of the isoallergens. Specific immunotherapy and diagnosis of conventional allergens are currently accomplished through the use of natural extracts of standard allergens which are subsequently formulated in the form of allergen vaccines. These aqueous vaccines are based on source materials of natural allergens, such as tree or herbal pollen, mite, hair and animal particle cultures. It is widely recognized that the composition of these natural source materials varies considerably depending on the time and place of collection of the same. Commercial allergen vaccines may, in addition, be formulated as allergen blends using various species. Knowledge of the composition of the extracts and the content of the essential allergens is a prerequisite for the reproducibility, safety and efficacy of the final product. The main challenge in the production of allergen vaccines is standardization, ie to ensure constant potency from batch to batch. Since the raw material is of natural origin, the variation is considerable and will have to be controlled through scientifically based measures. The extract composition should ideally reflect the composition of the water-soluble components of the allergen-like material as it is extracted from the surface of the airway mucosa and presented to the human immune system. However, all extracts contain various allergens that contribute to the binding of IgE in different combinations in patients. Ideally, therefore, all components would have to be controlled qualitatively and quantitatively, but with current technology this is not possible. Standardization is currently performed in several different ways, as each manufacturer has specific standardization procedures in their company. Standardization is carried out by techniques such as SDS-PAGE, also focusing on a series of immunoelectrophoretic techniques (ELISA) and ELISA using mono- and / or polyclonal antibodies, as well as radio-allergen (RAST) and other techniques related issues. An optimum batch standardization, such as the SQ standardization procedure, is essentially a three-step procedure: 1) to ensure a composition and constant rates across all components through semi-quantitative immunoelectrophoretic techniques, 2) to determine the components of the major allergens through quantitative immunoelectrophoresis, and 3) adjusting the total binding power of the IgE as determined by MagicLite® assays. In Europe, all standardization is currently performed for a particular reference preparation specific to a company, while in the US the FDA issues standards to be used by all manufacturers. All quantitative aspects of these current techniques depend on the antibodies as reagents and are therefore vulnerable and can change over time. 4 The absolute quantification of the specific components of the vaccine in complex mixtures of allergens is not straightforward and has not yet been defined as a routine, sensitive and high productivity technique.

Also in the food industry, routine and high productivity techniques are required for reliable detection and quantification of food allergens. Traces of nuts may be found in a food due to accidental cross-contamination during production. Companies that produce identical foods with and without, for example, dried fruit, face difficulties in cleaning production equipment between the production of different types of food. Traces of previously produced food, such as nuts, remain in the equipment. There are strong probabilities that the first lots of food produced without nuts using the same equipment contain traces of nuts. Foods that may cause an allergic reaction due to cross contamination of nuts or peanuts are, for example, chocolate, sweets, cookies, desserts, bonbons, donuts, cereals, smoothies, granola bars, muesli, pies, muffins, ice cream, sauce of barbecue. Cow's milk may cause an allergic reaction to small amounts of lactic proteins present in dairy products, cows' milk, cow's milk-based formulas or baby foods containing lactic proteins. To avoid contamination by lactic proteins during the production of baby foods or infant formulas for milk-allergic children, a method of detecting and quantifying lactic allergens is required. Reliable methods of detecting and quantifying food allergens are required to ensure compliance with food label specifications and to enhance consumer protection. Physico-chemical methods have already been described, for example, mass spectrometry as well as immunological methods. The common criteria of sensitivity, specificity, reproducibility, precision and accuracy have to be fulfilled. Nevertheless, some problems of cross-reactivity, matrix-related effects and food processing remain. Biological activity can be sustained even when the protein is denatured. Biological mass spectrometry (MS) was initially used to assess molecular weight and to identify proteins and peptides. In recent years, advances in mass spectrometry have resulted in techniques that can be used for the quantification of a number of biomolecules of complex mixtures, such as plasma, cell and tissue samples. Previous quantification techniques have defined only the relative quantification of proteins, while the more recent techniques evaluate the absolute amounts of the molecules in question. The rapid development of quantification techniques is essentially due to the progress in the area of proteomics, particularly in applications that distinguish, for example, healthy or disease states and identify the molecular markers of various diseases such as cancer, rheumatoid arthritis and disease of Alzheimer's disease. The main advantage of these MS quantification techniques is the high sensitivity of the techniques ranging from 300 amol to 300 fmol samples. WO 2004/070352 discloses a method for the quantification of peptides relative to an internal standard using isobaric labeling reagents or sets of isobaric labeling reagents. U.S. Patent No. 6872575 describes a method for identifying one or more proteins in samples of complex mixtures without purification of the protein or obtaining the peptidic signature of its compounds. U.S. Patent No. 6864089 describes a method of quantification using the differential isotopic labeling of peptide or protein samples.

Other methods of protein quantification using MS techniques are, for example, the AQUA technique using internal calibration peptides synthesized with stable (13C, 15N) isotopes to mimic the native peptides formed by enzymatic digestion using, for example, trypsin (Stemmann O. et al Cell 2001; 107 (6): 715-26, Gerber SA et al., Proc Natl Acad Sci USA 2003 100 (12): 6940-5).

Another method is the ICPL (" Isotope Coded Protein Labeling ") method described by Kellermann et al., Proteomics 5, 4-15, using, for example, 12C / 13C-nicotinic acid succinimide as a marker for ICPL. Mass spectrometry was first introduced in the field of allergology to characterize natural allergens including post-translational modifications, such as glycosylation patterns. It has also been used to characterize recombinant isolanergens and / or variants, many of which have been expressed in various expression systems such as Escherica Coll, Pichia Pastoris and Baculovirus expression systems.

Helsper et al., J. Allergy Clin. Immunol., Vol. 110, No. 1 (2002), pages 131-138, disclose the use of MS in the study of effective allergen isoforms identified by cloning in PCR and Swoboda et al., J. Biol. Chem., Vol. 270, no. 6 (1995), pages 2607-2613, liquid chromatography, MS and cDNA cloning are used to analyze the major allergen isoforms of birch pollen, Bet v 1.

In Kristiansson et al., RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Vol. 18, no. 14 (2004), pages 1592-1598, mass spectrometry is used to measure the amount of HHPA-adducted tryptic peptides from human serum albumin in nasal washes.

None of these references describes mass spectrometry for quantifying a group of allergen isoforms or homologous allergens in a sample, and none of these references describe the use of reference calibration peptides having an amino acid sequence identical to a constant amino acid sequence present in the group of allergens to be quantified.

Thus, a need still remains for a sensitive method by which active components such as allergens of the same or other species and / or isoallergens, for example, in a vaccine can be quantified. A method using MS techniques and allergen-specific and species specific amino acid sequences provides a very sensitive method by which the contents of groups of allergens (isoallergens or homologous allergens) can be quantified. The method according to the invention is useful, for example, in a release assay to ensure a safe and accurate amount of allergen during the production of a vaccine, in the final product and also during the various stages of storage of the active ingredients and / or products. The method is also beneficial in the development of vaccines against second generation allergens, for example, using recombinant allergens as active ingredients. Such a method would make it possible to optimize active ingredients in second generation allergen vaccines based on the knowledge and / or composition of the current vaccines.

SUMMARY OF THE INVENTION

According to the present invention, there is provided a method for the absolute quantification of allergens in a plurality of sources.

According to one aspect of the invention, there is provided a method for quantifying the absolute amount of allergen in an allergen sample, wherein the allergen is comprised of more than one homologous isoallergens or allergens, comprising the following steps: a) providing a known amount of one or more peptides for calibration with an amino acid sequence identical to a sequence found in the allergen quantitated by comparing the amino acid sequences of homologous isoallergens or allergens and preparing a synthetic allergen calibration peptide with this constant sequence and then labeling said allergen calibration peptide introducing mass modifying functionalities, b) decomposing the allergen sample to obtain a mixture of peptides, and optionally labeling such peptides with one or more labeling agents, introducing mass modifying functionalities, wherein, if the allergen degraded sample peptides and allergen calibration peptides are labeled, the labeling agent (s) used in the labeling of the allergen calibration peptides are different from the labeling agents used to label the peptides of the allergens. (c) quantify the absolute amount of allergen by correlating the amount of peptides for allergen calibration with the amount of the corresponding peptides of the allergen sample decomposed by mass spectrometry analysis.

In one variant of the invention, the allergen to be quantified consists of more than one isoallergen, for example, members of a group of allergens of the same species with more than> 67% amino acid sequence identity.

In another embodiment of the invention, the allergen to be quantified consists of more than one homologous allergen. According to the invention, the use of an amino acid sequence identical to a sequence to be found in the homologous isoallergens or allergens to be quantified is provided in the form of an allergen calibration peptide for absolute quantification and optionally allergen identification. Preferably, the decomposition of step b) results in a mixture of peptides wherein one of the peptides includes the same amino acid sequence as the allergen calibration peptide. 10

According to the invention, there is provided a method for obtaining an allergen calibration peptide for use in quantifying homologous isoallergens or allergens wherein the allergen calibration peptide is obtained by: identifying an amino acid sequence that is constant in the isoallergens or allergens to be quantified, by comparing the isoallergen or homologous allergen sequences, and preparing a synthetic allergen calibration peptide with this constant sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1. Alignment of the amino acid sequence of the mite allergen group 2 (Fig. 1a) and Bet v 1 (Fig. 1b), the major birch allergen, by Vector NTI software (Invitrogen). Putative amino acid sequences that can be evaluated as internal calibration peptides (useful for the quantification of isoallergens) are denoted in bold.

Fig. 2. Theoretical enzymatic cleavage by trypsin of allergens from domestic dust mites, a) Der f 2 and b) Der p 2; c) Phl p1, d) Phlp 5a, e) Phl p 5b and f) Bet v 1 (GPMAW, Lighthouse data). The specific peptides of the species selected for the quantification assays are indicated in bold and gray. 11

Fig. 3. a) Analysis of MALDI-TOF MS imprinting of a purified and natural Derf 2 and Der p 2 (1: 1) mixture of trypsin digested and b) MALDI -TOF MS imprinting analysis of a mixture of Der f 2 e

Purified and recombinant Der p 2 (1: 1) digested by trypsin.

Fig. 4. SDS-PAGE analysis of allergen extract of the dust mite separated by hydrophobic interaction chromatography. Fractions of the mite proteins were divided into two " pools " of proteins (I and II) and additionally subjected to quantification analysis.

Fig. 5. Sampling strategy using the chemical ITRAQ ™ (Applied Biosystems, Foster City, USA) in the quantification of allergens.

Fig. 6. MS analyzes of a) ITRAQ ™ labeled peptide 1, m / z 2353.44 (Der p 2, 32-48), b) peptide 2, m / z 2326.35 (Der f 2, 32-48) c ) peptides nDer p 2 digested by trypsin and labeled by ITRAQ ™ and (d) nFer 2 peptides.

Fig. 7. MS / MS fragmentation of the mixture of nDer f 2, nDer p 2, Peptide 1 and Peptide 2. The amount of isolerergens, nDer p 2 (114.10) and Der f 2 (115.10) in the sample of the mixture were calculated as a ratio between the signal area of m / z 114 and the area m / z 116 (Peptide 1) and ratio of the area m / z 115 to the area m / z 117 (Peptide 2 ). 12

Fig. 8. Reversed phase analysis of the mixture of nFer 2, nPer 2, Peptide 1 and Peptide 2 by SCX chromatography. MS and MS / MS analyzes were used to identify the peaks identified in the MALDI-TOF target.

Fig. 9. MS analysis of the trypsin-digested mixture of nBet v 1 and the internal calibration standard (AQUA peptide). The 6 Da mass difference between the native peptide and the calibration standard is shown in the upper corner of the figure.

DETAILED DESCRIPTION OF THE INVENTION

In the present context, the term " allergen " refers to any naturally occurring protein, modified protein, recombinant protein, mutant recombinant protein, or any protein fragment thereof, or mixtures of proteins with registries inducing allergic reactions, for example IgE-mediated through repeated exposure to a subject.

Examples of naturally occurring allergens include pollen allergens (tree pollen allergens, grass and grass), dust mite allergens (eg dust mites and storage mites), insect allergens (allergens from inhalation, saliva and venom), animal allergens from, for example, saliva, hair and particles from, for example, dog, cat, horse, mouse, etc., fungal allergens and food allergens.

Important pollens coming from trees, grasses and grasses are those that originate from taxonomic orders Fagales, Oleales, Pinales and plantanaceae, including, for example, birch, alder (Alnus), hazel (Corylus), carp Carpinus), olive (Olea), cedar (Cryptomeria and Juniperus), banana (Platanus), the order of Poales including, for example, herbs of the genus Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale and Sorghum and the orders of Asterales and Urticales, including, for example, herbs of the genus Ambrosia, Artemisia and Parletaria. Other important inhalation allergens are those derived from dust mites of the genus Dermatophagoides and Euroglyphus, from storage mites, for example Lepidoglyphus, Glycyphagus and Tyrophagus, those derived from cockroaches, mosquitoes and flies, e.g. Blatella, Periplaneta, Chironomus and Ctenocepphalides, and those derived from mammals such as cats, dogs and horses, venom allergens including those originating from the bite or insect bite such as those of the taxonomic order of Hymenoptera, including bees (superfamily Vespidea) and ants (superfamily Formicidae). Important inhalation allergens from fungi are, for example, those from the genus Alternaria, Cladosporium, Aspergillus and Penicillium. Examples of food allergens are wheat allergens (eg Tri a 18-19), crustaceans including shellfish (eg Met and 1, Pen a 1, Pen mie Pen m 2), shrimp, crab and lobster, fish ( for example, Gad c 1 and Sal s 1), chicken eggs (for example, Gal d 1, Gal d 2), peanut (for example, Ara h 1-8), soy (Gly m 1-4), milk (Bos d 4-8), nuts such as almond (Pru du 4), Brazil nut (Ber and 1, Ber and 2), cashew (Ana 1-3), hazelnut (for example, Cor a 1.04, Color a 2, Color a 8) and 14 walnut (for example Jug n 1-2, Jug r 1.3), celery (Api g 4, Api g 5), mustard (Sin ale Bra j 1) and sesame seeds (sets I-6), and in particular wheat allergens (for example Tri a 18-19), chicken eggs (for example, Gal d 1, Gal d 2), peanuts (for example, Ara h 1-8 ), soy (Gly m 1-4), cow's milk (Bos d 4-8).

Examples of recombinant allergens include, but are not limited to, plant pollen proteins / peptides, herb pollen, tree pollens, grass pollens, insect venom, dust mites and storage proteins, particulate matter saliva, fungal spores and food allergens (eg, peanuts, milk, gluten and eggs) prepared using recombinant techniques. Recombinant allergens can be obtained, for example, on a large scale using microbial expression systems that can be grown in fermenters, produced by recombinant DNA techniques, or chemical precursors when chemically synthesized. In one variant of the invention, the allergen is rBet v1, rAng g1, rCor a 1, rCar b1, rCry j1, rCry j2, role and 1, rAmb 1, rArt V 1, rCyn d 1, rDac g 1, rLol P 1, rLol P 5, rPhl P 1, rPhl P 5, rPoa P 1, rPoa P 5, rSor h 1, rDer f 1, rDer f 2, rDer P 1, rDer P 2, rEum m 1, rEur m 2, rGly d 1, rLe d 2, rBla g 1, rBla g 2, rFel d 1, rCan f 1, rCan f 2, rBos d 2, rEqu c 1, rEqu c 2, rMus m 1, rApi m 1, rApi m 2, rVes V 1, rVes V 2, rVes V 5, rDol m 1, rDol m 2, rDol m 5, rPol a 1, rPol a 2, rPol a 5, rAlt a 1 or rCla h 1 " i.rr " means recombinant).

A mutant recombinant allergen differs from " wild type " on the fact that the genes of the allergens have been modified by genetic manipulation methods, so that the polypeptides they encode exhibit substitutions, deletions and / or additions of one or more amino acids compared to " wild type ". Examples of mutant recombinant allergens include substitution variants, addition variants, oligomers, fragments, deletion variants, hybrid molecules and other variants.

Examples of modified allergens include allergens which in their natural form are associated with conditions of allergic diseases in susceptible individuals, said modified recombinant allergen being altered compared to the naturally occurring allergen. Included are allergen variants which contain some amino acid exchanges, allergen mutants, oligomers, fragments, deletion variants, hybrid molecules, myristylated, glycosylated, palmitolated and phosphorylated allergens and other variants. The modified allergen can be produced by any suitable method, such as the targeted mutagenesis method, a PCR method, chemical synthesis and a mixture of these methods.

In a variant of the invention, the allergen to be quantified is selected from one or more of the group consisting of Bet v 1, Aln g 1, Color ale Car b 1, Which a 1, Cry 1, Cry 2, Cup a 1, Cup s 1, Jun to 1, Jun to 2, Jun to 3, Ole and 1, Lig v 1, Syr v 1, Pia 1 1, Pia a 1, Pia a 2, Amb a 2, Amb t 5, Art v 1 , Art v 2, Art v 3, Par j 1, Par j 2, Par j 3, Sal k 1, Ave e 1, Cyn d 1, Cyn d 7, Dac g 1, Fes p 1, Hol 1 1, Lol p 1 and 5, Pha a 1, Pas η 1, Ph 1 p 1, Ph 1 p 2, Ph 1 p 3, Ph 1 p 4, Ph 1 p 5, Ph 1 p 6, Poa p 1, Poa p 5, Sec c 1, Sec c 5, Sor h 1, Der f 1, Der f 2, Der f 3, Der f 7, Der p 1, Der p 2, Der p 3, Der p 7, Der m 1, Eur m 1, Eur m 2 , Gly d 1, Gly d 2, Lep d 1, Lep d 2, Blo t 1, Tyr p 2, Bla g 1,16

Bla g 2, Per a 1, Per a 3, Per a 7, Fel d 1, Fel d 2, Fel d 3, Fel d 4, Can f 1, Can f 2, Bos d 2, Equ c 1, Equ c c 2, Equ c 3, Mus m 1, Rat η 1, Apis m 1, Apl m 1, Apl m 2, Ves v 1, Ves v 2, Ves v 5, Ves f 5, Ves g 5, Ves m 1, Ves m 2, Ves m 5, Ves p 5, Ves s 5, Ves vi 5, Dol m 1, Dol m 2, Dol m 5, Dol a 5, Pol a 1, Pol a 2, Pol a 5, Sol 1 1, Sol 1 2, Sol f 3 and Sol 1 4, Alt a 1, Alt a 3, Alt a 4, Alt a 5, Alt a 6, Cia h 1, Cia h 2, Cia h 6, Asp f 1, Bos d 4, Mal d 1, Mal d 3, Gly m 1, Gly m 2, Gly m 3, Ara h 1, Ara h 2, Ara h 3, Ara h 4, Ara h 5 or hybrids of any of these.

In another variant of the invention, the allergen to be quantified is one or more of the isoallergens selected from the group of herb pollen allergens as Phl p 1, Phl p 5, Phl p 6, Poa p 1, Poa p 5, Dac g 1, Fes p 1, Lol p 1 and Lol p 5, dust mite allergens as Der f 1, Der f 2, Der ρ 1 and Der p 2, venom allergens as Apl m 1, Apl m 2, Ves v 1, Ves v 2, Ves v 5, Dol m 1, Dol m 2, Dol m 5, Dol a 5, Pol a 1, Pol a 2 and Pol a 5, herb allergens as Amb a 1, Amb a 2, Par j 1, Par o 1 and Par m 1, birch allergens as Bet v 1, Japanese cedar allergens as Cry j 1 and Cry j 2, cockroach allergens as Per 1, olive allergens as Ole and 1, allergens cat allergens such as Canine allergens such as Can fie Can f 2, equine allergens such as Equ c Eq c 2, artemisia allergens such as Art v 1, Art v 2, Art v 3, mold allergens such as Alt a 1, Alt a 3, Alt a 4, Alt a 5, Alt a 6, Cia h 1, Cia h 2 and Cia h 6, and ant allergens. fire as Sol 1 2, Sol 1 3 and Sol 1 4.

In another embodiment of the invention, the allergen to be quantified is one or more of the isoallergens selected from the group consisting of grass pollen allergens such as 17

Phl p 1, Phl p 5 and Phl p 6, olive pollen allergens as Ole and 1, dust mite allergens as Der f 1, Der f 2, Der p 1 and Der p 2, venom allergens such as Ves v 1, Ves v 2 and Ves v 5, weed allergens such as Amb a 1, Amb a 2, Par j 1, Par ole Par m 1, and tree pollen allergens such as Bet v 1, Cry j 1 and Cry j 2.

In still another embodiment, the allergen to be quantified is one or more isoallergens selected from the group consisting of Der f 1, Der p 1, Der f 2 and Der p 2.

In yet another variant, the allergen to be quantified is one or more isoallergens selected from the group consisting of Phl p 1, Phl p 5, Phl p 6, Poa p 1, Poa p 5, Dac g 1, Fes p 1, Lol p 1, Lol p 5.

In yet another variant, the allergen to be quantified is one or more isoallergens selected from the group consisting of Amb.

An allergen of a single species may consist of several identical molecules. These similar molecules are called isoallergens when they share the following biochemical properties: a. similar molecular size; B. identical biological function, if known, for example, enzymatic action; and c. > 67% identity of the amino acid sequences. In the present context, members of a group of allergens having > 67% identity of the amino acid sequence and which are of the same species are designated as isoallergens. Each isoallergen can have multiple forms of fairly similar sequences differing in only a few amino acids; these are designated by variants and are encompassed in the term " isoallergen " in this context. 18

In the present context, the term " homologous allergens " refers to allergens of different species which are believed to share three-dimensional structures, molecular size, identical biological function, if known, for example, enzymatic action, and can share structural epitopes of the IgE antibodies. In another variant according to the invention, the homologous allergens contain > 20% identity of the amino acid sequences and share a common constant amino acid sequence of at least 2-20 amino acids, more preferably 4-15 and ideally 6-10. As an example of homologous allergens there may be mentioned, for example, Amp m 2 and Ves v 2, and Der f 2 and Der p 2.

In the present context, the term " allergen extract " refers to any extract obtained by extraction of an allergen source material as described in " Allergenic extracts ", H. Ipsen et al., Chapter 20, in Allergy, Principle and Practice (Ed. S. Manning) 1993, Mosby-Year Book, St. Louis. Such an extract may be obtained by aqueous extraction of water-soluble material followed by purification steps as filtration to obtain the solution, i.e. the extract. This may then be subjected to further purification and / or processing, such as lyophilization by removing substantially all of the water.

Generally, an allergen extract includes a mixture of proteins and other molecules. Allergen proteins are often classified as major allergen or, in an intermediate allergen, as minor allergen, or are not at all classified. An allergen extract generally includes larger and smaller allergens. Larger allergens usually constitute about 5-15% of a common allergen extract, more often about 10%. The classification of an allergen is based on an assessment of the clinical relevance of the allergen in question and is given below. Examples of major major allergens found in an extract include the allergens of groups 1, 5 and 6 (for example Phl p 1, 5 and 6), dust mite allergens of groups 1 and 2 (eg Der p 1, Der p 2), tree pollen allergen 1 (Bet v 1), cedar pollen allergens 1 and 2 (for example Cry1, Cry2), artemisia pollen allergens 1 and 2 (Amb a 1, Amb a 2), cat allergen 1 (ie, Fel d 1). The term " biological allergen source material " as used refers to any biological material that includes one or more allergens. Examples of such materials are PMB ("Pure Mite Body") or WMC ("Whole Mite Culture"), defatted and non-defatted pollens from, for example, grasses, grasses and trees, animal hair and particles, fur, mycelia and spores of fungi, bodies, venom or saliva of insects and food.

Materials of biological allergen origin may include materials such as external pollen and plant and flower residues from a source material of pollen allergens. The maximum level of contamination accepted with pollen from other species is 1%. It should also be totally free of waste flowers and plants, with a limit of 5% by weight. The term " allergen vaccine " as used in the present context includes at least one allergen originating from the same allergic source or originating from different allergenic sources, for example allergens from the group of herbs 1 and from the group of herbs 5, or allergens of mite group 1 and group 2 of mite species and 20 different herbs, respectively, weed antigens such as small and giant artemisia allergens, allergens of different fungi such as alternaria and cladosporium, allergens of trees such as birch, hazelnut , carp, oak and alder, food allergens such as peanut allergens, soy and milk. Vaccine preparation is generally known in the art. Vaccines are typically prepared in the form of

injections (liquid solutions or suspensions). Such a vaccine may be emulsified or formulated so as to allow nasal administration, as well as oral administration, including the non-oral and sublingual administrations. The immunogenic component in question may suitably be mixed with pharmaceutically acceptable excipients and compatible with the active ingredient. Examples of suitable excipients are water, serum, dextrose, glycerol and the like, as well as combinations thereof. The vaccine may further contain other substances, such as wetting agents, buffering agents or adjuvants which optimize the effectiveness of the vaccine.

According to one aspect of the invention, a method for quantifying the absolute amount of allergen in an allergen sample, wherein the allergen is comprised of more than one homologous isoallergens or allergens, comprises the following steps: a) providing a known amount of one or more allergen calibration peptides having an amino acid sequence identical to the sequence present in the allergen to be quantified, by identifying a constant 21 amino acid sequence within the allergen to be quantified by comparing the amino acid sequences of homologous isoallergens or allergens and preparing a synthetic allergen calibration peptide with this constant sequence and labeling said allergen calibration peptide by introducing mass modifying functionalities b) decomposing the allergen sample to obtain a mixture of peptides, and optionally labeling such peptides with one or more ag by introducing mass modifying functionalities wherein, if the allergen degraded sample peptides and the allergen calibration peptides are labeled, the labeling agent (s) used in the labeling of the calibration peptides of the allergens are different from the labeling agents used to label the peptides of the allergen sample decomposed, c) quantify the absolute amount of allergen by correlating the amount of peptides for allergen calibration with the amount of the corresponding peptides of the allergen sample decomposed by analysis by mass spectrometry.

In one embodiment of the invention, the calibration peptides and the peptides of the decomposed sample are labeled, but with different labeling agents.

In another embodiment of the invention, the calibration peptide (s) are labeled and the peptides from the decomposed sample are not labeled. 22

In a preferred embodiment of the invention as described above, a calibration peptide allergen is provided in step a). Thus, only one calibration peptide allergen is used for each allergen sample.

According to a preferred variant of the invention, the sample decomposed in step b) is, when labeled, labeled only with a labeling agent.

Thus, preferably, only one calibration peptide allergen is provided in step a) and, if labeled, the sample decomposed in step b) is labeled with only a labeling agent.

In addition, the calibration peptide is preferably labeled with only one labeling agent. Mass analysis, for example, MS or the like, may be carried out in mixtures of several pairs of an allergen sample and in an allergen calibration peptide according to the invention, for that particular allergen sample.

In the present context, the term " allergen calibration peptides having an amino acid sequence identical to the sequence present in the allergen to be quantified " refers to a constant region of the amino acid sequence, i.e., identical in the group of allergen isoallergens or in the homologous allergens to be quantified. According to a preferred embodiment of the invention, the calibration peptide (s) are selected so that the decomposition of allergen step b) (isoallergens or homologous allergens) to be quantified results in a mixture of peptides in which one of the peptides includes the same amino acid sequence as the allergen calibration peptide. Mass analysis, for example, MS or the like, may be carried out in mixtures of several pairs of an allergen sample and an allergen calibration peptide according to the invention, for that particular allergen sample. The number of amino acids in the allergen calibration peptide is preferably in the range of 2-20 amino acids, more preferably in the range of 4-15 amino acids and even more preferably in the range of 6-15 amino acids. The number depends on the optimum enzyme cleavage site compatible with the amino acid sequence of the sample, i.e., the constant or variable region sequence when the sample is cleaved by an enzyme. Furthermore, the allergen calibration standard to be used according to the invention depends on the labeling and the quantification method used, in order to emit a detectable signal and fragmentation when analyzed by an MS instrument.

In the present context, the term " allergen sample " refers to a sample which includes one or more allergens. In a variant of the invention, the allergen sample includes an allergen extract, a naturally occurring purified allergen, a modified allergen, a recombinant allergen, a recombinant mutant allergen, any allergen fragment, a mixture of isoallergens or mixtures of homologous allergens, or a combination thereof, and an allergen extract which includes " spiking " with purified or recombinant natural allergens. The allergen sample may take the form of a final product as an allergen vaccine in the form of a tablet or solution, or a product / intermediate withdrawn during production, such as after extraction of a source material or raw material, biological premium of allergens.

In a preferred embodiment of the invention, the allergen sample takes the form of an allergen extract, a final product in the form of a tablet or a product of an intermediate.

In one aspect of the invention, there is provided an allergen extract comprising a natural allergen and a recombinant allergen and which is obtained by quantifying the amount of allergen in a natural extract and the addition of recombinant allergen or purified natural allergen to the extract to obtain the amount required of the allergen in the final extract, as a natural extract subjected to " spiking " with purified or recombinant natural allergens. The method according to the invention makes it possible to quantify allergen (s) present in the form of isoallergens or allergens homologous and with a constant sequence of common amino acids of one or more species in a sample of allergens simultaneously or in one operation. It is possible to quantify the isoallergens of a species in a sample of allergens simultaneously or in a procedure using the method according to the invention.

Depending on the sample, it may be necessary to use denaturants and buffer solutions to obtain a suitable solution.

If the sample contains substances such as thiols, for example DTT or mercaptoethanol, high concentrations of detergents and / or denaturants such as SDS, octyl BD-glucopyranoside and Triton® X-100 and / or active proteases or primary amines (other than the allergen in which may interfere with the method according to the invention, the preparation of the sample may involve various treatments, for example, acetone precipitation. Alternative and / or denaturant buffers and detergents and substances which may interfere with the method according to the invention are listed, for example, in Applied Biosystems iTRAQ ™ Reagents Amine-Modifying Labeling Reagents for Multiplexed Relative and Absolute Protein Quantification Protocol, of Applied Biosystems, Foster City, CA, USA.

Depending on the complexity of the sample, it may be beneficial to pre-fractionate the sample prior to decomposition, for example if there are molecules to interfere with the detection of the allergen (s) in question in the sample. It may also be necessary to separate / elute the sample from its formulation and / or any adjuvant, for example, aluminum hydroxide or calcium phosphate. To obtain a less complex mixture, the sample may be fractionated through the use of various chromatography techniques, such as hydrophobic interaction, ion exchange and / or immunoaffinity chromatography.

In one embodiment of the invention, the allergen sample is a fraction resulting from pre-fractionation, for example, an allergen extract fraction which includes one or more isoallergens.

In one variant of the invention, the allergen sample is a fraction from the pre-fractionation step in which the sample was fractionated according to the size, solubility, electrical charge and / or specificity of ligands. In another embodiment of the invention, pre-fractionation is accomplished by chromatography, such as hydrophobic interaction chromatography, ion exchange chromatography, size exclusion chromatography or affinity chromatography, for example, hydrophobic interaction chromatography.

An example of pre-fractionation of an intermediate product containing Group 1 and Group 2 HDM allergens by the use of hydrophobic interaction chromatography is shown in Fig. 4. The fractions containing, respectively, Group 1 and 2 HDM allergens are separated by their physicochemical properties and identified by immunoprecipitation. Both fractions can then be subjected to quantification studies.

In one embodiment of the invention, the allergen sample is desalted after chromatography.

In one embodiment of the invention, the allergen sample is reduced and any cysteine residue is blocked prior to decomposition, such as, for example, alkylation.

According to the invention, the sample is broken down by treatment with one or more enzymes to obtain a mixture of peptides. The enzyme may be selected to have a predictable decomposition pattern that permits identification and quantification of the peptides obtained by comparison with the allergen calibration peptide. The enzyme may be one or more proteases such as, for example, two proteases or one or more distinct enzymes. Examples of proteolytic enzymes include trypsin, papain, pepsin, ArgC, LysC, V8 protease, AspN, pronase, chymotrypsin and carboxypeptidase C. For example, proteolytic trypsin ezimate is a serum protease that cleaves the peptide bonds between lysine or arginine and an unspecified amino acid to thereby produce peptides which include an amino terminal amino acid (N-terminal) and a carboxy (C-terminal) terminal amino acid of lysine or arginine. In this way, protein cleavage peptides are predictable and their presence and / or amount in a sample of a trypsin digestion is indicative of the presence and / or amount of the protein of its origin. In addition, the free amino termini of a peptide may be a good nucleophile which facilitates its labeling. Since the activity of the enzymes is predictable, the sequence of the peptides produced can be predicted by the decomposition of a protein of a known sequence. With this information, " theoretical " information can be generated " about the peptide. A determination of the " theoretical " peptide fragments ", for example, in a computer-aided analysis of the son fragment ions of mass spectrometric analysis of an actual sample may thus be used to identify one or more peptides.

In one embodiment of the invention, the allergen sample is decomposed prior to labeling by digestion of the sample with at least one proteolytic enzyme to partially or totally decompose the sample. In another embodiment of the invention, the proteolytic enzyme is selected from the group consisting of trypsin, papain, pepsin, ArgC, LysC, V8 protease, AspN, pronase, chymotrypsin and carboxypeptide C, or a combination thereof, for example, as selected between the group consisting of ArgC, LysC and trypsin, or a combination thereof. In yet another embodiment of the invention, the enzyme is trypsin. The digested sample may be prepared prior to labeling by any of several methods, if necessary. 28

For those skilled in the art, it will be apparent that there are numerous possibilities for labeling the sample and for allergen calibration peptides, to introduce, in a predetermined manner, different mass modifying functionalities that enable quantification of the peptides of the allergens. The label may, for example, be made as described in WO 2004/070352, US 6864089, in Stemmann O. et al., Cell 2001; 107 (6): 715-26, and Gerber S.A. et al., Proc. Natl. Acad. Sei USA 2003; 100 (12): 6940-5.

In one embodiment of the invention, labeling is performed according to iTRAQ ™ technology (Applied Biosystems, Foster City, CA, USA).

In accordance with this variant of the invention, the labeling of the allergen decomposed sample and / or the calibration peptide is performed by a set of isometric or isobaric labeling reagents such as the iTRAQ ™ Reagents (Applied Biosystems, Foster City, CA, USA) . Each of these reagents contains a reactive group (RG) that reacts with the analyte, and a single reporter group (RP) that produces a " signature ion " in MS / MS analysis. Accordingly, labeling the decomposed allergen sample yields an analyte named sample RP-X-LK-Y. Analysis of the analyte is performed by adjusting a mass spectrometer so that X and Y join the fragment. Fragmentation of the X-bond releases the reporter from the analyte and the reporter can then be determined independently of the analyte. Fragmentation of the Y-bond releases the RP-LK combination of the analyte. Thus, based on fragmentation, the presence and / or amount of the reporter can be correlated with the presence and / or amount of the analyte in the sample. Labeling with, for example, 4 iTRAQ ™ reagents enables simultaneous simultaneous quantification of different allergen samples (allergen samples are decomposed and each peptide mixture is labeled with different iTRAQ ™ reagents). The possibility of simultaneous analysis of different allergen samples allows the comparison of the labeled peptides, the sample (s), with the known amount of allergen calibration peptide (s) and, therefore, enables quantification and identification through MS / MS in one step. Labeling of the samples using iTRAQ ™ reagents and / or other labeling reagents may be performed according to the manufacturer's procedure, as described in Fig. 5.

Other methods of protein quantification using MS techniques are, for example, the AQUA technique which uses internal calibration peptides synthesized with stable (13C, 1SN) isotopes to mimic the native peptides formed by enzymatic digestion using, for example, trypsin ( Stemmann 0. et al Cell 2001; 107 (6): 715-26, Gerber SA et al., Proc Natl Acad Sci USA 2003 100 (12): 6940-5).

Another method is the ICPL (" Isotope Coded Protein Labeling ") method described by Kellermann et al., Proteomics 5, 4-15, using, for example, 12C / 13C-nicotinic acid succinimide as a marker for ICPL.

In a variant of the invention, the distinctly labeled peptide (s) and the allergen calibration peptide (s) are labeled separately and mixed after labeling prior to quantitation. 30

Depending on how the allergen and calibration peptide labeling is performed, an appropriate form of identification may be selected.

In a variant of the invention, the allergen is positively identified by comparing the labeled peptide (s) of the allergen and the calibration peptide (s) by peptide identification analysis. Ion exchange chromatography may also be used for the separation of the peptides in combination with reverse phase chromatography as two-dimensional chromatography, and, if necessary, for reducing the amount of any salts and organic compounds prior to MS analysis.

In one variant of the invention, quantification is performed using mass spectrometry.

In another embodiment of the invention, the identification of allergen and / or isoallergens can be performed using tandem mass spectrometers and other mass spectrometers capable of selecting and fragmenting molecular ions. This is especially suitable when iTRAQ ™ reagents are used in the labeling.

Tandem mass spectrometers (and single-phase mass spectrometers) have the ability to select and fragment molecular ions according to their mass-to-charge ratio (m / z) and then record the spectrum of the resulting sonic fragment (son) . More specifically, son ion fragment spectra can be generated by subjecting selected ions to dissociative energy levels (for example, collision-induced dissociation (CID)). For example, the ions corresponding to the labeled peptides of a given ratio m / z can be selected in a first mass analysis, fragmented and reanalyzed in a second mass analysis. Representative instruments that can perform such tandem mass analysis include, but are not limited to, four-sector magnetic mass spectrometers, time-of-flight tandem mass spectrometers, triple quadrupole mass spectrometers, quadrupole mass spectrometers , " ion-trap " type mass spectrometers and quadrupole mass spectrometers for hybrid flight time (Q-TOF).

These mass spectrometers can be used in conjunction with a number of ionization sources, including, but not limited to, electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI). Ionization sources can be used to generate charged species for the first mass analysis, where the analyzes do not yet have a fixed charge. Mass spectrometry instruments and additional fragmentation methods include post-source decomposition in MALDI-MS and high energy CID instruments using MALDI-TOF (flight time) -TOF MS. For a recent study of mass spectrometers, see R. Aebersold and D. Goodlett, Mass Spectrometry in Proteomics. Chem. Rev. 101: 269-295 (2001). See also U.S. Patent No. 6319476 for a discussion of TOF-TOF mass analysis techniques. The allergen calibration peptide (s) (the variable or constant sequence) are chosen depending on whether the quantification is performed relative to an allergen or homologous allergens or allergens or specific isoallergens). 32

In one embodiment of the invention, absolute quantification of an allergen (absolute amount of allergen isoallergens) can be performed.

Using the iTRAQ ™ reagents, the chosen allergen calibration peptide is labeled with an isomeric or isobaric marker of the set of labels, for example iTRAQ-114, iTRAQ-115, iTRAQ-116 or iTRAQ-117, used to label the peptides of the allergen.

Thus, in a variant of the invention, labeling is done with iTRAQ-Ι14, iTRAQ-115, iTRAQ-116 and / or iTRAQ-117.

As soon as the relative amount of reporter for the allergen calibration peptide, or reference peptides, is determined relative to the relative amounts of the reporter for distinctly labeled peptides, it is possible to calculate the absolute amount (often expressed in concentration and / or quantity ) of all the distinctly labeled peptides in the sample mixture and hence calculating the amount of allergen (for example, the absolute amount of isoallergen (s) of a species when the sample is an extract). The acquisition of MS and MS / MS from samples labeled by iTRAQ ™ may be performed, for example, using the 4700 Explorer ™ software. In addition, the GPS Explorer software can also be used to perform searches in the database that would result in the definitive identification of the peptides from MS / MS. The data obtained can then be used for quantification based on the known amount of allergen calibration peptide, i.e., the ratio of the sample to the allergen calibration peptide. 33

In a variant of the invention (i), the allergen to be quantified is Der f 2 and the allergen calibration peptide includes amino acids 32-48 of Der f 2, or (ii) the allergen to be quantified is Der p 2 and the calibration peptide of allergens includes amino acids 32-48 of Der p 2. The method according to the invention is useful, for example, in a release assay, to ensure a safe and predictable amount of allergen during the production of a vaccine, and in the final product , and also during the various stages of storage of ingredients and / or products, and crude extracts. The method according to the invention is useful in the development of second generation allergen vaccines, for example using recombinant allergen as active ingredients, optimizing the active ingredients in second generation allergen vaccines based on the knowledge and / or composition of the present vaccines. The present vaccines are often formulated using allergens of various species, and the method would also be beneficial in determining the composition of the allergens specific for a species of such allergen mixtures. The method according to the invention may be used in validation, in which traces of allergens are measured, in release assays and in intermediate and final product analyzes.

Peptides that can be used as calibration peptides can be produced through the use of protein and / or necleotide databases and cleavage analysis programs and / or in vitro mass-printing experiments.

In the present context, the term " allergen calibration peptide " refers to a calibration standard of 34 allergens with an amino acid sequence identical to the constant sequence of the group of isoallergens or homologous allergens. The allergen calibration peptide is preferably prepared by peptide synthesis.

In some cases, the sequence of the allergen in question is known. An official list of allergens can be found, for example, on the website (www.allergen.com), which is maintained by I.U.I.S. Allergen Nomenclature Sub-committee. Known allergen sequences can be obtained from protein and nucleotide databases, for example, Uniprot Knowlegdebase. Protein and / or nucleotide sequences can be searched using, for example, Sequence Retrieval System (SRS) or using keywords, for example by submitting the entry name (ID), description (DE), gene name (GN) , species (OS) and / or organelle (OG). Further analysis of the protein / allergen in question is performed using, for example Vector NTI (Invitrogen) software and / or by using the Proteinomics server ExPASy (Expert Protein Analysis System) of the Swiss Institute of Bioinformatics (SIB). Alignment of allergen isoform sequences and existing allergen species is performed using, for example, the Blast search, which can be used to align homologous protein and / or nucleotide sequences. Sequence alignments provide a way of comparing, for example, novel sequences with previously characterized genes and / or proteins. Alignment sequences of allergens or homologous isoallergens can be used to demonstrate the identical (constant) and variable sequences in and between allergen species, as shown in Fig. 1.

In order to obtain optimum calibration peptides, a cleavage analysis of the allergen in question can be simulated using cleavage (decomposition) analysis programs. Allergen sequences may be subjected, for example, to the GPMAW program (Ughthouse data, Odense, Denmark), designed to support MS analyzes. Analysis of the cleavage (decomposition) of an allergen sequence can be deduced from various proteases known as trypsin, Asp-N and Lys-C and / or a combination of two or more. The resulting theoretical peptides (Fig. 2) are then used to check optimal decomposition enzymes and define the synthetic peptides that can be used as calibration peptides.

On the other hand, calibration peptides can be deduced from in vitro mass printing experiments, in which the allergens are cleaved by enzymes and mixed. Species-specific peptides can be detected by mass-print analysis and identified by searching in databases, for example using the Mascot search engine, as described below:

Der f 2 and Der p 2 natural (n) purified and Der f 2 (A61501) and Der p 2 (BAA01241) (r) can be dissolved in 25 mm Tris-Cl, pH 7.5, 1.0 M urea . An aliquot of recombinant molecules (rDer f 2 and rDer p 2) and natural molecules (nDer f 2 and nDer p 2) may be mixed (for example, 1: 1) or digested as individual allergens by trypsin and subsequently mixed. The mixed and / or individual allergen digestions are desalted and evaluated by mass print analysis. Species-specific peptides can be identified by mass-print analysis from the blending of these two species. Individual digested HDM 2 allergens can be mixed after trypsin digestion, and the same species-specific constant sequences can be demonstrated. Based on the constant species-specific sequences, the synthetic peptides can be designed. Quantification and verification of the peptide sequences can be performed by labeling the individual allergens and the calibration peptides, for example with iTRAQ ™ reagents and analyzed by tandem mass spectrometry (MS / MS). Natural allergen extracts, for example, an intermediate product of two species HDM Dermatophagoides farinae and Dermatophagoides pteronyssinus (ALK-Abelló, H0rsholm, Denmark), (1.0 mg / ml dry weight) are diluted. To remove the interfering component, samples may be precipitated with acetone. The precipitate containing proteins can then be resolved into the chosen buffer. For quantification purposes, the sample (s) and the synthetic peptides selected from the two species used as the calibration peptide may be labeled with iTRAQ ™ reagents and analyzed by MS / MS. The final product, for example powder mite allergen tablet, is dissolved in 20 mm Na phosphate buffer, pH 7.0. To remove the interfering components, the sample must be pre-fractionated and / or precipitated with acetone. The dissolved tablet and chosen calibration peptides are labeled with iTRAQ ™ reagents and analyzed by tandem MS / MS. 37

Liberation study. The final product, for example, blend of 5 herbs, including 5 species of herbs coupled with aluminum hydroxide, is dissolved in a certain buffer which can elute bound aluminum hydroxide allergens. Unbound allergens may then be separated and / or desalted into a given digestion buffer and the chosen calibration peptides may be labeled, for example, with iTRAQ ™ reagents and quantification may be assessed by MS / MS.

EXAMPLES

Example 1

Identification of single constant regions of Der f 2, Der p 2, Ph 1 p 1, Ph 1 p 5 and Bet v 1 and synthetic calibration peptides Absolute quantification of isoallergens using the single constant sequences, ie, the Der 2, Der p 2, Phl p 1, Phl p 5 and natural Bet v 1 has been demonstrated through the use of two distinct approaches. The two calibration peptides were synthesized to evaluate the iTRAQ ™ labeling technique (Applied Biosystems). In addition, four calibration peptides were synthesized to evaluate the stable isotope labeling technique, such as Protein-AQUA ™ (Sigma-Aldrich).

Calibration peptides with species-specific sequences corresponding to the amino acid sequence of 32-48 in Der f 2 and Der p 2, 149-158 in Phl p 1, 123-135 in Phl 38 p 5a, 115-127 in Phl p 5b and 151-164 in Bet v 1, were designed based on the amino acid sequence alignments (Vector NTI) (Fig. 1), GPMAW cleavage analyzes (Fig. 2) and Blast database searches. In vitro mass-mediated analyzes of natural allergens digested by Derf 2, Der p 2, Der p 2, Phl p 1, Ph 1 p 5, Bet v 1 and Der f 2 and Der p 2 blends were performed in vitro to demonstrate the occurrence of the peptides species. All samples and mixtures digested in vitro were identified using the Mascot search engine (Matrix Science Inc., MA, USA).

Synthetic peptides corresponding to amino acid sequence 32-48 in Der f 2 and Der p 2 were obtained from GENOSYS, Sigma, Texas, USA. The concentration of the peptides was determined by amino acid analyzes (Sigma GENOSYS, Texas, USA). Stable isotope-labeled synthetic peptides (protein-AQUA ™ peptides) corresponding to amino acid sequence 149-158 in Phl p 1 (Arg 13 C 15 N), 123-135 in Phl p 5a (Arg 13 C 15 N), 115-127 in Phl p 5b a (Arg 13 C 15 N) and 151-164 in Bet v 1 (Val 13 C 15 N) were obtained from Sigma GENOSYS, Texas, USA (Table 1).

Example 2

Purification of natural and recombinant allergens of group 2 of herbs, birch and mites

Natural Der p 2 and Der p 2 were purified from 100 mg extracts of Dermatophagoides farinae and Dermatophagoides pteronyssinus (ALK-Abelló, H0rsholm, 39

Denmark). Phl p 1 and 5 were purified from 50 mg Phantan prtalense extract (ALK-Abelló) and natural Bet v 1 from 50 mg birch extract verrucose (ALK-Abelló). Purification of the molecules was performed as described in the literature (Johannessen BR et al., FEBS Lett. 2005, 579: 1208-12, Aasmul-Olsen, S. et al., New Horizons in Allergy Immunotherapy, edited by Sehon et al., Plenum New York 1996, pp. 261-65, Petersen A. et al., Clin Exp. Allergy, 1994, Mar 24 (3): 250-6, Ipsen H. & Lowenstein HJ Allergy Clin Immunol 1983; 72 (2): 150-59). Der f 2 and Der p 2 were expressed in the Pichia pastoris expression system and purified as described in the literature (Johannessen BR et al., FEBS Lett. 2005; 579: 1208-12). Proteins were stored as lyophilized aliquots at -20 ° C. The concentration of the purified allergens was measured using the extinction coefficient of one of the A2o isoallergens and using a Lambda 800 UV / VIS spectrometer (Perkin Elmer Instruments, CA, USA).

Example 3

Pre-fractionation of HDM extracts and protein digestions

Hydrophobic interaction chromatography was used to pre-fractionate the extracts of Dermatophagoides farinae (Der f) and Dermatophagoides pteronyssinus (Der p). Fractionation of the mite extracts was performed with a 1.0 ml HiTrap Phenyl column (GE-Healthcare, Uppsala, Sweden). The column was equilibrated with 50 mm of 40

Na-phosphate buffer (Merck), pH 7.0 and volumes using a linear gradient decreasing. Chromatography was performed separately for each of the extracts of HDM, Der f and Der p. The fractions were analyzed by SDS-PAGE (Invitrogen, Carlsbad, CA, USA) and based on these analyzes, the HDM proteins were divided into two " pools " of proteins (Fig. 4). &Quot; pools " of Der f and Der p containing HDM 2 allergens were subjected to a dialysis step against 10 mm of ammonium bicarbonate (BDH, Poole, England). &Quot; pools " dialyzates of Der f and Der p were lyophilized, divided into aliquots of ~ 5 mg (dry weight) in vials and stored cold at -20 ° C. The aliquots of Der f and Der p containing HDM 2 allergens were additionally submitted to absolute quantification studies.

Example 4

Enzymatic cleavage and labeling with iTRAQ ™

The labeling with iTRAQ ™ was used in the three sample sets: a) 15æg natural Der f 2 and 15æg natural Der p 2 b) 15æg recombinant Der f 2 and 15æg recombinant Der p 2 and c) 100 pg of extracts Der fe Der p.

The synthetic peptides, 15æg of Peptide 1 (Der p 2) and 15æg of Peptide 2 (Der f 2) were dissolved in 100 mm of triethyl ammonium bicarbonate (TEAB) pH 8.5 and labeled 41 for use as calibration standards for each of the sets of experiments as described below. Cleavage of cysteine-free residues, enzymatic cleavage using trypsin and labeling with iTRAQ ™ reagents were carried out according to the manufacturers' protocol:

Each of the six samples and the two internal calibration standards were dissolved in 20 μΐ of 100 mm TEAB, pH 8.5. The protein samples were denatured with 1.0 μΐ of 0.05% SDS and reduced by 2.0 μΐ of 4.8 mM Tris (2-carboxyethyl) phosphine TCEP at 60 ° C after blocking the cysteine residues by 1.0 μΐ of 10 mm s-methyl methanethiosulfonate (MMTS) at room temperature. Protein samples were digested with 10% (w / w) modified trypsin (Promega, Madison, WI, USA) for 18 hours at 37 ° C. The iTRAQ ™ label of the trypsin digested samples and the internal calibration peptides, Peptides 1 and 2, was performed at room temperature. Each labeling reagent iTRAQ from 114 to 117 was dissolved in 70 μΐ of 70% ethanol, which was then applied under the sample (s). The final volumes of the reagent mixtures were 100 μΐ / sample. The labeled samples were stored at -20 ° C.

The peptides digested by natural Der p 2 trypsin, Der p 2 recombinant and Der p extract were labeled with iTRAQ-114. Trypsin-digested peptides of native Der f 2, recombinant Der f 2 and Der f extract were labeled with iTRAQ-115. Synthetic peptides, Peptide 1 (Der p 2) and Peptide 2 (Der f 2) were labeled with iTRAQ-116 (Der p 2) and iTRAQ-117 (Der f 2) (Fig 5). Each of the 42 labeled samples was analyzed by Voyager STR and / or 4700 Proteomic Analyzer (Applied Biosystems) to demonstrate labeling of the peptides. Fragment analyzes were performed by MS / MS with 4700 Proteomic Analyzer (Applied Biosystems). The labeled samples were diluted 1:10 and 10 μΐ of the sample (s) were desiccated with C18 microcolumns (Zip Tips, Milipore) and / or manual C18 microcolumns (Poros R2, Applied Biosystems). The sample was eluted on the target with 1.0 μΐ of 70% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA), and a 1.0 μ matriz matrix of alpha-cyano-4-hydroxy (CHCA) (Agilent Technologies, Böblingen, Germany) above the sample. The sample was dried and subjected to MS analysis.

MS analyzes of internal calibration standards revealed masses corresponding to Peptide 1 with m / z 2353.44 and Peptide 2 with m / z 2326.35 (Figs. 6a and 6b). The results show that modification in Der p 2 specific Peptide 1 and Der f 2 specific Peptide 2 corresponded to modification by iTRAQ ™ reagent when bound to the amino-terminal and C-terminal lysine. MS analyzes of iTRAQ ™ -labeled Der p 2 natural and recombinant peptides revealed iTRAQ ™ modified masses with m / z 2353.29. Likewise, MS analyzes of the labeled natural and recombinant Der f 2 peptides revealed peptides modified by iTRAQ ™ with m / z 2326.29, respectively. No cleavages were observed by trypsin. These results demonstrate that iTRAQ-labeled Peptides 1 and 2 can be used as internal calibration standards for absolute quantification assays of Der p 2 and Der f 2 isoallergens.

Example 5

Absolute quantification of the mixture of two different species of HDM Absolute quantification of the isoallergens of the species Dermatophagoid.es farinae and Dermatophagoides pteronyssinus was first used in the direct mixing of the purified natural allergens. Derivatives of iTRAQ ™ -labeled tryp Derivatives were mixed with Peptides 1 and 2 in a ratio of 1: 1: 1: 1 diluted in 1: 5 and 1:10 and desalted by C18 manual microcolumns (Poros R2 , Applied Biosystems). The sample was eluted on the target using 10 μΐ of 5æg / μΐ CHCA (Sigma) in 70% ACN (Sigma), 0.1% TFA (Fluka). Fragment analyzes were performed by MS / MS through 4700 Proteomic Analyzer (Applied Biosystems).

Analyzes of the m / z 2353.29 fragments revealed am / z 114 signals at / z 116, corresponding to the natural Der p 2 reporter ions and Peptide 1. The amount of naturally occurring Der p 2 isoallergens in the sample was calculated as the ratio of the m / z signal area 114 to the m / z area 116, the internal calibration pattern (Fig. 7a). Fragment analyzes by MS / MS of m / z 2326.29 showed am / z 115 signals at / z 117, corresponding to the natural Der f 2 reporter ions and Peptide 2. The amount of natural Der f 2 isoallergens at sample was calculated as the ratio between the area of the m / z signal 115 and the area of m / z 117, the internal calibration standard (Fig. 7b). In addition to the absolute quantification of the peak lists of the ionic fragments of m / z 2353,29 and m / z 2326,29, 44 were subjected to analysis of databases using the Mascot search engine (Matrix Science). Analyzes to the database resulted in the identification of the peptides as HDM 32-48 allergens of Dermatophagoides farinae and Dermatophagoides pteronyssinus.

Example 6

Separation of the labeled peptide mixtures by two-dimensional chromatography

Absolute quantification of the mixture of recombinant Der f 2 and Der p 2 and the isoallergens in the complex mixture of extracts of Der f and Der p by two-dimensional chromatography was evaluated. Cation exchange chromatography (SCX) was evaluated as the first dimension separation step and reverse phase chromatography as a second dimension separation step.

Der p 2 and Der f 2 were mixed with Peptides 1 and 2 in a ratio of 1: 1: 1: 1, in a final volume of 50 μΐ. The extracts of Der f and Der p and Peptides 1 and 2 were mixed in a ratio of 4: 4: 1: 1, in a final volume of 50 μΐ.

Separation of the labeled peptide mixtures using cation exchange chromatography (recombinant Der p 2 and Der f 2 and extracts of Der p 2 and Der f 2);

Sample mixtures were diluted 1:10 in 5% ACN (Sigma), 0.05% formic acid (Merck) and subjected to SCX. SCX was run on a 0.8x50 mm Zorbax BIO-SCX (3.5 μιτι) column (Agilent Technologies) in a SMART ™ system (GE-45

Healthcare, Uppsala, Sweden). The column was equilibrated with 5% ACN (Sigma), and 0.05% formic acid (Merck) and chromatographed with a linear gradient increasing from 0 to 100% 5% ACN (Sigma), 0.05% formic acid (Merck) and 0.05 M NaCl (Merck) for 30 minutes. The flow velocity was 50 μΐ / min and the chromatography was monitored at 214 nm. 50 μl fractions were collected and 1.0 μ of each fraction was analyzed by MS with Voyager-STR (Applied Biosystems) and / or 4700 Proteomic Analyzer (Applied Biosystems). Peptides at m / z 2353.29 and m / z 2326.29 were observed to elute at the end of the gradient along with some other HDM peptides. The fractions of the Der f and Der p mixture (extracts) containing the peptides at m / z 2353.29 and m / z 2326, 29 were selected to be further separated by reverse phase chromatography, see infra. However, the quantification of the mixture of rDer f 2 and rDer p 2 (recombinant) and the internal calibration standards was performed directly after SCX of the target plate.

Analyzes of the MS / MS fragments of m / z 2353.29 revealed am / z 114 signals in Î »116 corresponding to the reporter ions of Der p 2 and Peptide 1. The amount of Der p 2 clone of the rDer p 2 / rDer f 2 was calculated as the ratio of the signal area m / z 114 to the m / z area 116, the internal calibration standard. MS / MS fragments analyzes of m / z 2326.29 showed signals at m / z 115 in / z 117 corresponding to the reporter ions of Der f 2 and Peptide 2. The amount of Der f 2 clone of the rDer p 2 / rDer f 2 was calculated as the ratio between the signal area m / z 115 and the m / z area 117, the internal calibration standard. The peak lists of the ionic fragments of m / z 2353.29 and m / z 2326.29 were subjected to analysis of databases using the Mascot search engine (Matrix Science). Analyzes to the database resulted in the identification of the peptides as HDM 32-48 allergens of Dermatophagoides farinae and Dermatophagoides pteronyssinus.

The peptides at m / z 2353.29 in m / z 2326.29 in both samples, the mixture of recombinant Der f 2 and Der p 2 and the extracts of the Der fe Der p mixtures eluted on SCX column with similar retention times . This experiment showed that SCX can be used as a step of fractionation and desalination in simpler mixing samples prior to the quantification of the allergens. Experiments have also shown that SCX can be performed as a first-step fractionation step for more complex allergen mixtures analyzes, such as the conventional allergen extracts used in immunotherapy.

Separation of labeled peptides (from extracts of Der p 2 and Der f 2) by SCX followed by reverse phase chromatography and followed by absolute quantification by MS MALDI-TOF-TOF

The iTRAQ ™ labeled HDM peptides were separated from the SCX fractionation as described above with PepMap C18 column (3 pm) (LC Packings Dionex, Sunny Vale, CA, USA). The column was equilibrated with 0.05% TFA (Fluka), 2% ACN (Sigma). The peptides were eluted with 0.04% TFA (Fluka), 80% ACN in a 0-50% gradient over 80 min, 50-100% in 120 min. Chromatography is performed with Ultimate300 (LC Packings, Dionex), 20 μΐ / min and monitrized at 47 to 210 and 214 nm. 1.0 μΐ of the SCX fraction was injected into the column. Fractions were collected by direct detection on a MALDI-TOF target plate. Detection was performed with the Probot instruments (LC Packings, Dionex), connected to an Ultimate 3000 instrument online. Detection was performed every 30 sec. mixing the HCCA matrix (Agilent Technologies) with the sample at a ratio of 1: 1.

Detected samples were analyzed by MS and MS / MS using the 4700 Proteomic Analyzer (Applied Biosystems). The peptides at m / z 2353.29 and m / z 2326.29 were identified at the target sites and were found to correspond to the signals at 214 nm in the chromatography (Fig. 8). Analysis of the MS / MS fragments of m / z 2353.29 revealed that the am / z 114 signals in / z 116 corresponded to the reporter ions of Der p 2 and Peptide 1. The amount of Der p 2 isoallergen in the mixture of extracts of Der p / f was calculated as the ratio between the signal area of m / z 114 and the m / z area 116, the internal calibration standard. Fragment analyzes by MS / MS of m / z 2326.29 revealed that the am / z 115 signals in / z 117 corresponded to the reporter ions of Der f 2 and Peptide 2. The amount of Der f 2 isoallergen in the mixture of extracts of Der p / f was calculated as the ratio between the area of the m / z signal 115 and the m / z area 117, the internal calibration standard. The peak lists of the ionic fragments of m / z 2353.29 and m / z 2326.29 were subjected to analysis of databases using the Mascot search engine (Matrix Science). Analyzes to the database resulted in the identification of the peptides as HDM 32-48 allergens of Dermatophagoides farinae and Dermatophagoides pteronyssinus. 48

This experiment shows that reverse phase chromatography can be used as the second dimension of the step of fractionating samples of single and / or complex mixtures of allergen extracts for quantification of the isoallergens.

Example 7

Absolute quantification of isoallergens using the AQUA strategy

In the AQUA technique (Stemmann 0. et al., Cell 2001; 107 (6): 715-26, Gerber SA et al., Proc. Natl Acad Sci USA 2003; 100 (12): 6940-5), the peptides (13C, 15N) are synthesized to mimic the native peptides formed by enzymatic digestion using, for example, trypsin. Incorporation of an isotope labeled amino acid residue alters the peptide molecular masses typically between 6 and 10 Da. Unlike the iTRAQ technique, the internal calibration samples or standards have to be modified by the labeling reagents. In quantitation experiments, for example, when using LC-MS / MS, the abundance of a specific ionic fragment of the native peptide sample and the internal calibration standard synthesized can be measured as a function of the retention time in phase chromatography reverse. Absolute quantification is determined by comparing the abundance of the known internal standard with the native peptide sample.

Synthetic internal calibration peptides of natural Phl p 1, Phl p 5 a and b, and Bet v 1 were designed as described above (Fig. 2). Synthetic peptides are described more fully in Table 1.

Table 1: Marking of synthetic internal calibration standards

Species Allergen Amino acid sequence Theoretical mass Modification Modified mass Beetle Bet v 1 AVESYLLAHSDYN 1552,73 (Go 13c 1558.64 verrucosa 15N) Phleum Phl P i SAGEVEIQFR 1135,57 (Arg 13c 1145,27 prantense 15N) Phleum Phl P YDAYVATLSEALR 1471 , 74 (Arg 13c 1481.66 prantense 5 a 15N) Phleum Phl P FDSFVASLTEALR 1455.74 (Arg 13c 1465.66 prantense 5b 15N)

Phl p 1, Phl p 5 and Bet v 1 were dissolved in 25 mm Tris-Cl (Sigma), 1.0 M urea (Fluka) pH 7.8 at a concentration of 2.5 pmol / μ. Internal calibration standards were mixed in samples in a ratio of 1: 1. Digestion was carried out by 10% trypsin (Promega) at 37 ° C for 18 hours. The samples were stored at -20 " C. Phl p 1, tryptic digested, Phl p 5 and Bet v 1 were analyzed by MS and MS / MS using Voyager STR and / or 4700 Proteomic Analyzer (Applied Biosystems). All samples were diluted 1:10 in 0.1% TFA (Fluka) and 1.0 μ in each of the samples was desalted by manual C18 microcolumns (Poros R2, Applied Biosystems). 50

MS analyzes of trypsin-digested natural Phl p1 revealed the native peptide at m / z 1135.60 and the internal calibration peptide of isoallergens from Phl pia m / z 1145.61 (data not shown). MS / MS analyzes of the internal calibration peptide showed a fragmentation pattern that corresponded to the unique amino acid sequence of the natural Phl p 1 isoallergens.

MS analyzes of wild-type trypsin-digested Phl p 5 revealed that the native Phl p 5 peptide at m / z 1471.81 and the native Phl p 5b peptide at m / z 1455.77 (data not shown). The internal calibration peptides of Phl p 5a and Phl p 5b at m / z 1481.83 and at m / z 1465.78. MS / MS analyzes of the internal calibration peptides showed fragmentation patterns that corresponded to the single amino acid sequences of the Phl p 5a and Phl p 5b isoallergens.

MS analyzes of trypsin-digested natural Trypsin 1 revealed the native peptide at m / z 1552.76 and the internal calibration peptide of Bet isoallergens via m / z 1558.78 (Fig. 9).

The MS / MS analyzes of the internal calibration peptide showed a fragmentation pattern that corresponded to the unique amino acid sequence of the natural isoallergens of Bet v 1.

For the absolute quantification of isoallergens of Bet v 1,

Phl p 1, Phl p 5a and Phl p 5b, samples may be subjected to analysis of, for example, LC-coupled MS / MS instruments such as LCQ DecaXP (ThermoFinnigan), QSTAR® hybrid LC / MS / MS system, and LC / MS / MS 4000 Q TRAP system (Applied Biosystems). 51

Experiments with the AQUA peptides have shown that stable isotope-labeled synthetic peptides that mimic the native isoallergen sequences can be used for the absolute quantification of natural Phl p 1, Phl p 5 and Bet v 1 isoallergens. In addition, the analyzes of the databases of tryptic digests did not reveal any cleavages lost by trypsin. Two-dimensional chromatography of the combination of SCX and RP-HPLC as described for the iTRAQ chemistry may be used in the fractionation of more complex allergen blends of chemically identical native and synthetic internal calibration standards. 52

REFERENCES REFERRED TO IN THE DESCRIPTION The present list of references cited by the applicant is presented merely for reasons of convenience to the reader. It is not part of the European patent. Although care has been taken during the compilation of references, it is not possible to exclude the existence of errors or omissions, for which the IEP assumes no responsibility.

Patents of the invention cited in the description • WO 2004070352 A • US 6872575 B • US 6864089 B • US 6319476 B [0011]

Non-patent literature cited in the description • Stemmann O. et al. Cell, 2001, vol. 107 (6), 715-26 Gerber S.A. et al. Proc. Natl. Acad. Know. USA, 2003, vol. 100 (12), 6940-5 • Kellermann et al. Proteomics, vol. 5, 4-15 [0014] • Helsper et al. J. Allergy Clin. Immunol., 2002, vol. 110, 131-138 Swoboda et al. J. Biol. Chem., 1995, vol. 270 (6), 2607-2613 Gerber S.A. et al. Proc. Natl. Acad. Know. USA, 2003, vol. 100 (12), 6940-5 [53] Kristiansson et al. RAPID COMMUNICATIONS IN MASS SPECTROMETRY, 2004, vol. 18 (14), 1592-1598 [0017] • R. Aebersold; D. Goodlett. Mass Spectrometry in Proteomics Chem. Rev., 2001, vol. 101, 269-295 Johannessen B.R. et al. FEBS Lett., 2005, vol. 579, 1208-12 [0111] Aasmul-Olsen S. et al. New Horizons in Allergy

Immunotherapy, Plenum Press, 1996, 261-65 [Petersen A. et al. Clin. Exp. Allergy, March 1994, vol. 24 (3), 250-6 [0111] • Ipsen H .; Lowenstein H. J. Allergy Clin. Immunol. 1983, vol. 72 (2), 150-59 [0111]

Lisbon, 9/6/2010

Claims (18)

A method of quantifying the absolute amount of allergen in an allergen sample, wherein the allergen is comprised of more than one homologous isoallergens or allergens, the following steps comprising: a) providing a known amount of one or more peptides allergen calibration standard with an amino acid sequence identical to the sequence present in the allergen to be quantified by identifying a constant amino acid sequence in the allergen to be quantified by comparing the amino acid sequences of homologous isoallergens or allergens and preparing an allergen calibration peptide synthesized with this constant sequence and labeling said allergen calibration peptide by introducing mass modifying functionalities b) degrading the allergen sample to obtain a mixture of peptides, and optionally labeling such peptides with one or more labeling agents, by introducing mass modifying functionalities wherein, if the allergen degraded sample peptides and the standard allergen calibration peptides are labeled, the labeling agent (s) used in the labeling of the standard allergen calibration peptides are different from the labeling agents used to label the peptides of the degraded allergen sample, c) quantify the absolute amount of allergen by correlating the amount of standard peptides to the allergen calibration with the amount of the corresponding peptides from the degraded allergen sample by analysis by mass spectrometry. A method according to claim 1, wherein the allergen sample is degraded so as to obtain a mixture of peptides, and the labeling of the peptides obtained with one or more labeling agents is carried out by introducing modifying functionalities and the absolute amount of allergen is quantitated by correlating the amount of standardized allergen calibration standard peptide with the amount of corresponding labeled peptides of the degraded allergen sample using mass spectrometry. A method according to claim 1, wherein the allergen sample is degraded so as to obtain a mixture of peptides, and the absolute amount of allergen is quantitated by correlating the amount of standard allergen calibration standard peptides labeled with amount of corresponding labeled peptides from the degraded allergen sample, using mass spectrometry. A method according to any one of claims 1-3, wherein the standard allergen calibration peptides have an amino acid sequence identical to the amino acid sequence in a peptide obtained by degradation according to step b). A method according to claims 1-4, wherein the allergen to be quantified consists of more than one isoallergen. A method according to claims 1-4, wherein the allergen to be quantified consists of more than one homologous allergen. A method according to any one of claims 1-6, wherein the allergen to be quantified is one or more isoallergens selected from the group consisting of Phl p 1, Phl p 5, Phl p 6, Poa p 1, Po p p 5 , Dac g 1, Fes p 1, Lol p 1, Lol p 5, Der f 1, Der f 2, Der p 1, Der p 2, Api m 1, Api m 2, Ves v 1, Ves v 2, Ves v 5, Dol m 1, Dol m 2, Dol m 5, Dol a 5, Pol a 1, Pol a 2, Pol a 5 Amb a 1, Amb a 2, Par j 1, Par or 1, Par m 1, Bet v 1, Cry j 1, Cry j 2, Per a 1, Ole and 1, Fel d 1, Can f 1, Can f 2, Equ c 1, Equ c 2, Art v 1, Art v 2, Art v 3, Alt a 1, Alt a 3, Alt a 4, Alt a 5, Alt a 6, Cia h 1, Cia h 2, Cia h 6, Sol i 2, Sol i 3 and Sol i 4. A method according to claim 7, wherein the allergen to be quantified is one of the isoallergens selected from the group consisting of Phl p 1, Phl p 5, Phl p 6, Ole and 1, Der f 1, Der f 2 , Der p 1, Der p 2, Ves v 1, Ves v 2, Ves v 5, Amb a 1, Amb a 2, Par j 1, Par m 1, Bet v 1, Cry j 1 and Cry j 2. A method according to claim 8, wherein the allergen to be quantified is one or more isoallergens selected from the group consisting of Der f 1, Der p 1, Der f 2 and Der p 2 4. A method according to claim 7, wherein the allergen to be quantified is one or more isoallergens selected from the group consisting of Phl p 1, Phl p 5, Phl p 6, Poa p 1, Poa p 5, Dac g 1 , Fes P 1, Lol p 1 and Lol p 5. A method according to claim 7, wherein the allergen to be quantified is one or more isoallergens selected from 2. group consisting of Amb a i 1 e Amb a A method according to any one of the preceding claims, wherein the labeling is performed with the iTRAQ ™ technique. A method according to claim 12, wherein the labeling is with iTRAQ 114, iTRAQ 115, iTRAQ 116 and / or iTRAQ 117. A method according to any one of the preceding claims, wherein (i) the allergen to be quantified is Der f 2 and the standard allergen calibration peptide includes amino acids 32-48 of Der f 2, or (ii) the allergen to be quantified is Der p 2 and the standard peptide of allergen calibration includes amino acids 32-48 of Der p 2. A method according to any one of the preceding claims, wherein the allergen is positively identified by comparing the mixture of the allergen peptides with the standard allergen calibration peptides by peptide identification analysis. 5 A method according to any one of the preceding claims, wherein the allergen sample is degraded by digestion with at least one proteolytic enzyme to partially or totally degrade the sample. A method according to claim 16, wherein the proteolytic enzyme is selected from the group consisting of trypsin, papain, pepsin, Arg C, Lys C, protease V8, Asp N, pronase, chymotrypsin and carboxypeptidase C, or a combination of the same. A method according to claim 17, wherein the enzyme is trypsin. Lisbon, 9/6/2010